We have developed an in vitro system whereby we can study the molecular mechanism underlying activation of the ATR kinase signaling cascade in response to DNA alkylation. ATR is a member of the phosphoinositide 3-kinase-related kinase (PIKK) family involved in DNA damage signaling. We have shown that MutSalpha and MutLalpha recruit ATR to sites of cytoxic O6-methylguanine-thymidine mispairs resulting in activation of the ATR kinase activity and phosphorylation of cell cycle checkpoint proteins such as Chk1 and SMC1. ATR-dependent cell killing mediate by p53-dependent pathways contributes to the cytotoxicity of DNA alkylating agents. Modified base analogs commonly used as adjuvant therapy for colorectal cancer such as fluorouracil are currently being examined to see if they also elicit MMR-dependent cell killing.